Abnormalities in microbiota/butyrate/FFAR3 signaling in aging gut impair brain function

Aging-related abnormalities in gut microbiota are associated with cognitive decline, depression, and anxiety, but underlying mechanisms remain unstudied. Here, our study demonstrated that transplanting old gut microbiota to young mice induced inflammation in the gut and brain coupled with cognitive decline, depression, and anxiety. We observed diminished mucin formation and increased gut permeability (“leaky gut”) with a reduction in beneficial metabolites like butyrate because of decline in butyrate-producing bacteria in the aged gut microbiota. This led to suppressed expression of butyrate receptors, free fatty acid receptors 2 and 3 (FFAR2/3). Administering butyrate alleviated inflammation, restored mucin expression and gut barriers, and corrected brain dysfunction. Furthermore, young mice with intestine-specific loss of FFAR2/3 exhibited gut and brain abnormalities akin to those in older mice. Our results demonstrate that reduced butyrate-producing bacteria in aged gut microbiota result in low butyrate levels and reduced FFAR2/3 signaling, leading to suppressed mucin formation that increases gut permeability, inflammation, and brain abnormalities. These findings underscore the significance of butyrate-FFAR2/3 agonism as a potential strategy to mitigate aged gut microbiota–induced detrimental effects on gut and brain health in older adults.


Mishra et al Supplementary Figure S2
Supplementary Figure S3.Schematic presentation of the fecal microbiome transplantation (FMT) experiment and the schedules for young to young, old to young, old to old, and young to old, along with the assays performed.

Mishra et al Supplementary Figure S3
Mishra et al Supplementary   Mishra et al Supplementary Figure S12 CMT

Inflammation in Colon
The expression of inflammatory genes (Il1b, Il6, and Tnfa) significantly increased in the colon of old mice compared to young mice.All values represent the mean of 5-10 animals in each group, and error bars represent the standard error of means.Statistical significance was determined using a t-test, and the p-value ***p<0.001 is statistically significant.
. (A,B) The levels of systemic markers of leaky gut (LBP and sCD14) significantly increased in 10-12 weeks old mice who received old FMT compared to those who received young FMT.(C) The expression of inflammatory genes (Il1b, Il6, and Tnfa) significantly increased in old FMT recipients compared to young FMT recipients.(D) Similarly, the old FMT recipient mice showed higher systemic inflammatory markers like IL6 and TNFα compared to the levels in young FMT recipients.All values represent the mean of 5-10 animals in each group, and error bars represent the standard error of the means.Statistical significance was determined using a t-test and/or ANOVA, with a p-value of ***p<0.001,indicating statistical significance. .(A) Volcano graph of differentially expressed genes in the intestines of old and young mice.(B) Muc2 expression in the ileum of old and young donor mice.(C,D) Small intestine histology (H&E staining) shows fewer goblet cells (white bubble-like structures indicated by red arrows) in old mice than in young mice.(E) Fecal mucin content in older mice decreased compared to young mice.(F) The goblet cell population in the colon was found to be lower in older mice than in younger mice, and old FMT recipients also showed decreased goblet cells compared to young FMT recipients.Number of goblet cells per villi was measured by FIJI.All values represent the mean of 5-10 animals in each group, and the pictures are representative of 5-10 animals.Five pictures were taken of each animal's tissue sections.Statistical significance was determined using a t-test, and the p-value ***p<0.001 is statistically significant.
. (A-E) Multivariate analyses correlating butyrate with Muc2 (A), buk (B), and but (C) genes as well as Muc2 expression with buk and but genes (D,E) in young donors, old donors, young FMT recipients, and old FMT recipients.Analyses are done using Pearson correlation analysis and r 2 values plotted using r-scripts.Values represent the mean of 3-4 independent replicates in cells cultures for each group, and error bars represent the standard error of means.Statistical analyses were performed using t-test and/or ANOVA, as applicable, and the actual p-values are presented in the graphs.Butyrate treatment increased PAS positive (PAS + ) area percentage (%) quantification in CMT93 cells dampened by old FCM treatment measured by FIJI.Values represent the mean of 3-4 independent replicates in cells cultures for each group, and error bars represent the standard error of means.Statistical analyses were performed using t-test and p-values ***p<0.001are statistically significant.
Schematic of group randomization, gut cleansing, FMTs, treatments, and assays conducted to test the effects of butyrate in Villin-Cre driven intestine specific Ffar2 (iF2) and Ffar3 (iF3) knockout (KO) mice compared to their wildtypes (WT), as data presented in main Figure7A-M.
The expression of inflammatory markers (Il1b, Il6, Tnfa) (A-C) significantly increased in the colon of 7-months old intestine-specific Ffar2 (iF2) and Ffar3 (iF3) knockout (KO) mice compared to their ageand sex-matched wildtype (WT) controls.All values represent the mean of 5-10 animals in each group, and error bars represent the standard error of the means.Statistical significance was determined using a t-test, as applicable, and p-values of *p<0.05,**p<0.01,and ***p<0.001are statistically significant.

et al Supplementary Figure S10 Measures in Organoid Measures in CMT93 Cell Line
donor Supplementary FigureS7.Hierarchical differential clustering analyses reveal significant differences in metabolites between old and young gut (ileum).All the values represent the mean of 5-10 animals in each group, and error bars represent the standard error of means.Statistical significance was determined using t-test.Mishra et al SupplementaryFigure S8 Supplementary Figure S8.Comparing the abundance of three SCFAs-(A) Butyrate, (B) Acetate, and (C) Propionate -in old to young feces shows the greatest decrease (based on p-values) in butyrate.Values represent the mean of 5-10 animals in each group, and error bars represent the standard error of means.Statistical significance was determined using t-test, and the actual p-values are presented in the graphs.Mishra et al Supplementary Figure S9 Supplementary Figure S9.Heatmap of Pearson correlation values among fecal metabolites with mucin genes (Muc2, Muc6, Muc13), tight junction protein genes (Zo1, Ocln1), leaky-gut markers (FITC-dextran permeability [indicated as "FITC"], and inflammation (both intestinal [indicated by "i"] and systemic [serum IL6 and TNFα]) revealed unique clustering.The highest correlation was between butyrate and Muc2 gene expression.cells and organoid cultures for each group, and error bars represent the standard error of means.Statistical analyses were performed using one-and/or two-way ANOVAs, and p-values *p<0.05,**p<0.01,and ***p<0.001are statistically significant.

Table S2 .
RFA (Randon forest analysis) of intestinal specific marker gene expression in mice administration with Young vs Old FMT (fecal microbiome transplant)